6 alpha-fluoro-16 alpha-hydroxy-1, 4-pregnadienes



United States Patent ()fi ice 2,838,546 Patented June 10, 1958 6a-FLUORO-16a-HYDROXY-1,4-PREGNADIENES No Drawing. Application February 19, 1958 Serial N0. 716,005

9 Claims. (Cl. 260-39145) '16',21-diesters thereof, the ll-keto analogues and the 16,21-

diesters'thereof, and a method for the production thereof. The novel compounds of this invention are illustratively represented by the following formula:

wherein Y is selected from the group consisting of hydrogen and fluorine, X is selected from the group consisting of the carbonyl radical C=O) and the p-hydroxymethylene radical and R is selected from the group consisting of hydrogen and .the acyl radical of an organic carboxylic acid,preferably a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

{The new compounds, l-dehydro-6 z-fiuoro-l6a-hydroxyhydrocortisone and 1-dehydro-6a,9a-difluoro 161x hydroxyhydrocortisone, their 16,21-diesters and the ll-keto analogues and the esters therof, are highly active adrenocortical hormones having greater glucocorticoid and antiinfiammatory activity than hydrocortisone or cortisone. In addition these compounds have diuretic activity and have salt-losing properties which make them especially well suited in the management of chronic congestive heart failure and in the treatment of cirrhosis of the liver, the nephrotic and adrenogenital syndromes and the treatment of eclampsia and preeclampsia.

The novel l-dehydro-6a-fluoro-l6a-hydroxyhydrocortisone, 1-dehydro-6a,9a-difluoro-16a-hydroxyhydrocortisone, the 16,21-diesters thereof, the ll-keto analogues and the l6,21-diesters thereof can be given in oral, parenteral or topical compositions. The compounds can be administered to the animal organism in conventional dosage forms such as pills, tablets and capsules for oral use or in conventional liquid forms as are used with natural and synthetic cortical steroid hormones for injection use. For topical use they can be administered in the form of ointments, creams, lotions and the like with or without coacting antibiotics, germicides and the like.

The process of the present invention comprises microbiological hydroxylation of l-dehydro-6a-fluorohydrocortisone and 1-dehydro-60,9a-difiuorohydrocortisone to pro duce the corresponding 16-hydroxy compounds. Esterification of the thus-produced 16-hydroxylated compounds is productive of the 16,21-diesters. The corresponding ll-keto analogues of these l6-hydroxylated compounds are obtained by oxidation of the ll-hydroxyl group of the above 16,21-diester compounds with an oxidation agent such as chromic acid. If the free alcohols of the ll-keto compounds are desired, the additional step of hydrolyzing the 16,21-diesters, such as with an alkali metal base, is necessary.

Starting materials for the present invention are l-dehyrohydrocortisone, prepared as shown in the preparations.

In the bioconversion step of the present invention, the operational conditions and reaction procedure and details maybe those already known in the art of steroid bioconversion as illustrated by the Murray et -al., U. S. Patent 2,602,769, issued July 8, 1952, utilizing, however, the action of an organism of the genus Streptomyces. Among the species which are useful in the fermentation step of the present invention are Streptomyces roseochromogenus (Waksman Collection No. 3689), Streptomyces sp. (A. T. C. C. 11009), and Streptomyces roseochromogenus (A. T. C. C. 3347) The selected species of actinomycete is grown on a medium suitably containing assimilable non-steroidal carbon, illustratively carbohydrates, such as dextrose, assimilable nitrogen, illustratively soluble or insoluble proteins, peptones or amino acids; and mineral constituents,

illustratively sodium or ammonium phosphate and magnesium sulfate The medium may desirably have a pH before inoculation of between about 6.5 to about 7.8, though a higher or lower pH may be used. A pH of between about 6.8 and about 7.4 is preferred for the growth of actinomycetes and a temperature range from about twenty to about 35 degrees Centigrade with about twenty to 32 degrees centigrade preferred.

The growth period required before the steroid to be fermented is exposed to the actinomycete does not appear to be critical, for example, the steroid may be added either before sterilization of the medium, at the time of inoculating the medium or at sometime later, for example, 24! to 48 hours later. The addition of steroid substrate to be fermented may be accomplished in any suitable manner, such as by dispersing the steroid substrate, either alone with a dispersing agent, or in solution in an organic solvent. Either submerged or surface culture procedures may be used with facility, although submerged culture is preferred.

The temperature during the period of fermentation of the steroid may be the same as that found suitable for the growth of the organism. It need be maintained only within such range as supports life, active growth, or the enzyme activity of the Streptomycete.

The time required for the fermentation of the steroid varies somewhat with the procedure. When the steroid is added to the actinomycete after substantial growth of the organism, for example, after sixteen to 24 hours at optimum temperature, the conversion of steroid substrate begins immediately and is substantially complete in from two to ten days, five days being generallysatisfactoryf After completion of the steroid fermentation, the resulting transformed steroid is recovered from the fermentation reaction mixture by extracting the fermentation reaction mixture, including the fermentation liquor and mycelium with an organic solvent for steroids, for example, methyl isopropyl ket'one, methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichlomethylene, ether, amyl acetate, benzene, and the like.

The fermentation liquor and mycelium may be separated and then separately'extracted with suitable solvents. The

extracts can be combined, either before or after Washing with an alkaline solution, illustratively sodium bicarbonate, suitably dried, as for example, over anhydrous sodium sulfate, and the resulting purified transformed steroid obtained by recrystallization from organic solvents, by trituration or by chromatography in order to isolate the thus-obtained steroids from the other transformation products.

Bioconversion of 1-dehydro-6a-fluorohydrocortisone and 1-dehydro-6a,9a difluorohydrocortisone, according to the fermentation procedure above described, is productive of 6a-fiuo ro-115,16,17a,2l-tetrahydroxy-lA-preg nadiene-3,20-dione and 604,9ot-dlflllOI'O-llfi,l6a,l7a,2ltetrahydroxy-1,4-pregnadiene-3,20-dione, respectively.

The 16-hydroxylated compounds thus produced can be esterified to produce the corresponding 16,2l-diesters. This reaction can be performed under esterification conditions known in the art, e. g., by the reaction of the 'hydroxy compound with the selected acid halide, e. g., acid chloride or acid bromide, the anhydride of a hydrocarbon carboxylic acid, or by reaction with the selected acid, in the presence of an esterification catalyst or with an ester-under ester exchange reaction conditions. Reaction conditions which are apt to afiect the labile 11B- hydroxy group or 6-fluoro group should be avoided.

Compounds thus produced include the 16,21-diacyloxy -2,6-, 3,4- and 3,5-dimethy1benzoic ethylbenzoic, 2,4,6-

trimethylbenzoic, 2,4,6-triethylbenzoic, a-napthoic, 3- methyl-u-naphthoic, an aralkyl acid, e. g., phenylacetic, phenylpropionic, diphenylacetic, triphenylacetic, an unsaturated acid, e. g., acrylic, maleic, vinyl acetic, propiolic, undecolic, etc. Illustrative of the esters thus produced are the 16,21-diacylates such as 6oc-fluoro-11p,16u, 17a,21-tetrahydroxy-1,4-pregnadiene 3,20 dione 16,21- diacetate, 60,9a difluoro-llfl,16a,17ot,21-tetrahydroxy- 1,4-pregnadiene-3,20-dione 16,21-diacetate, and the like.

The llfl-hydroxyl of the thus-produced 16,21-diester compounds can be oxidized to the corresponding 11- ketone with an oxidizing agent. Oxidizing agents such as chromic acid, potassium dichromate, a haloamide, and the like are operative. The oxidation can be carried out by a variety of methods, such as for example, by oxidizing the said 11/3-hydroxy steroid in acetic acid-water solution with chromium trioxide, using molar quantities or a slight excess, such as from ten to thirty percent excess, or by oxidizing with a haloamide or imide of an acid, such as N-bromoacetamide, N-chlorosuccinimide, or N-bromosuccinimide dissolved in pyridine, dioxane, or other suitable solvents. At the conclusion of the desired oxidation reaction, the excess oxidant is generally destroyed by addition of methyl alcohol, ethyl alcohol, and

the like for the chromic acid oxidant or a bisulfite for N-bromoacetamide, N-bromosuccinimide and other N- haloacylamides and imides. Thereafter, the resulting 11- keto product is recovered by conventional means, such as by dilution with water and extraction with a Water-immiscible solvent, e. g., methylene chloride, ether, benzene, toluene, ethyl acetate, or the like. Illustrative of the ll-keto 16,21-diesters thus produced are, for example, 6u-fluoro-16a,17a,21-trihydroxy 1,4 pregnadiene- 4- trihydroxy-1,4-pregnadiene-3,11,20 7 trione 16,21-diacetate, and the like.

The ll-keto 16,21-diest'ers thus produced may, if desired, be converted to the free alcohols, i. e., Got-fluoro- 16m,17a,21-trihydroxy-1,4-pregnadiene 3,11,20 trione and 606,9(X. difluoro 16o't,17a,21 trihydroxy-lA-pregnadiene 3,11,20 trione. The alcohols are obtained from the acylates by hydrolysis in accordance with general hydrolysis procedures known in' the art. A preferred procedure is to employ an alkali-metal bicarbonate in a substantially oxygen-free solution of a mixture of a lower alkanol and water. The hydrolysis reaction is carried out at a temperature between ten and thirty degrees centigrade while protecting the mixture from atmospheric oxygen. After the hydrolysis is complete, the reaction mixture is neutralized with an acid, e. g., acetic acid, and the hydrolyzed product recovered from the reaction mixture by evaporation and crystallization, extraction with methylene chloride, or the like. The 16,21-diesters, when desired, can again be prepared by esterification of the hydroxyls by esterification procedures hereinbefore described. The preferred diesters are those derived from an organic carboxylic acid, preferably a hydrocar'bon carboxylic acid containing from one to twelve carbon atoms, inclusive.

The following preparations and examples are illustrative of the process and products of the present invention, but are not to be construed as limiting.

PREPARATION 1 The 3-ethylene ketal of methyl 3,1I-diketo-Safien-oxido- 1 7(20) [cisl -pregnen-21-oate To a solution of 5.0 grams of the 3-ethylene ketal of methyl 3,11 diketo-4,l7(20)-[cisl-pregnadien-2l-oate, prepared in the manner described in U. S. Patent 2,707,184, in milliliters of chloroform Was added a chilled solution of 1.9 grams of perbenzoic acid dissolved in 31.5 milliliters of chloroform. The solution was maintained at about four degrees centigrade for 24 hours, and then at room temperature for 72 hours. The solution was then washed with a five percent aqueous solution of sodium bicarbonate and then with water. The chloroform layer was separated, dried and the solvent distilled to give a residue of 5.3 grams of solid. Crystallization of this solid from methanol gave 2.24 grams of product melting at to degrees centigrade and after two crystallizations from methanol, there was obtained pure 3-ethylene ketal of methyl 3,ll dlket0-5a,6c.-

-' oxido-17(20)-[cis]-pregnen-21-oate melting at 206 to 209 degrees centigrade having an [cab of plus 37 degrees (CHCl and having the analysis given below:

Analysis.-Calculated for C H O C, 69.20; H, 7.75 Found: C, 69.59; H, 7.81.

PREPARATION 2 Methyl 3,11diketo-5u-hydroxy-6 8-fluoro-l 7(20 allopregnen-Zl-oate To a solution of 1.73 grams of 3-ethylene ketal of methyl 3,11 diketo 51,6 oxido 17(20) [cislpregnen-Zl-oate in sixteen milliliters of methylene chloride was added six milliliters of 48 percent hydrofiuon'c acid. The heterogeneous mixture was stirred for two hours, made slightly basic with 300 milliliters of five percent sodium bicarbonate solution, and extracted with methylene chloride. The extract was washed, dried and evaporated to dryness to give 1.62 grams of crude solid. Chromatography gave two fractions: A, 481 milligrams eluted with methylene chloride plus five percent acetone and B, 921 milligrams eluted with methylene chloride plus ten and twenty percent acetone. Crystallization of fraction A from acetone-Skellysolve B hexanes gave 390 milligrams of methyl 3,11-diketo-5u-hydroxy-6B-fiuoro- 17(20)-allopregnen 21-oate, melting point 254 to 260 degrees centigrate. to 263 degrees Centigrade.

Methyl 3,1 1 -diket-5a-hydroxy-6fl-fluorO-1 7 (20) -all0- pregnen-ZZ -0ate S-ethylene ketal A mixture of 1.9 grams of methyl 3,11-diketo-ahydroxy 6e lluoro 17(20) allopregnen 21 oate, 59 milligrams of p-toluenesulfonic acid mouohydrate and. 31 milliliters of distilled ethylene glycol was added to 800 milliliters of benzene. The mixture was stirred and refluxed for two hours, with the condensate passing through a water trap to remove the water. After reflux the mixture was cooled, washed with water and evaporated to dryness to give a crude solid which on recrystallization from aoetone-Skellysolve l3 hexanes gave 1.96 grams of methyl 3,11 diketo 5a hydroxy 65 fluoro- 17 (20) allopregnen- 21 oate 3-ethlene .ketal, melting point 170 to 173 degrees centigrade.

PREPARATION 4 To a solution of 1.96 grams of methyl 3,11-dik6t0-5ozhydroxy-6l3-fluoro- 17 (20) -allopregnen-2 l-oate 3 -ethylcne ketal in 850 milliliters of anhydrous ether was added 3.7 grams of lithium aluminum hydride and the mixture was stirred for a period of one hour. Two hundred milliliters of water was added slowly and the ether phase separated. The aqueous phase was extracted with ethyl acetate and the extracts added to the ether phase. The

grees centigrade. I

PREPARATION 5 The acetate was prepared by allowing 0.87 gram of l 5a,11p,21 trihydroxy 6e flu-oro 17(20) allopregnen- '3-one 3-ethy1ene-lcetal to stand overnight in tcnmilliliters of acetic anhydride and ten milliliters :of pyridine. The solution was then poured into ice water to give 0.92 gram of 5a,l1B-dihydroxy 6B fluoro 21 acetoxy-17(20)- allopregnen-ione 3-ethy1ene ketal, melting point 140 to 150 degrees centigrade, which on recrystallization from acetone-Skeliysolve B hexanes gave 0.77. gram, melting point 149 to 153 degrees Centigrade.

PREPARATION 6 3,20-a'z'one .S-ethyl'eIw ketal To a solution of 0.77 gram of SuJIfi-dihydroxy-lSflfluoro 21 acetoxy-17(20)-allopregnen-3-one 3 ethyIene ketal in 35 milliliters of tertiary butyl alcohol was added A one milliliter of pylidine, 1.9 milliliters of 1.95 molar tertiary butyl alcohol solution of N-methylmorpholine oxide peroxide, and 13.1 milligrams of. osmium tetroxide (9.1 milliliters of tertiary 'butyl alcohol solution containing 1.4 1- milligrams 050 per milliliter). The solution was stirred for a period of 2.5 hours, fifteen milliliters of five percent sodium hydrosulfite added, stirred for an additional ten minutes, 0.7 gram or" finely ground synthetic magnesium silicate added, stirred for period of twenty minutes more and filtered. The filtrate was taken to dryness under reduced pressure (below fifty degrees centigrade) and the residue dissolved in methylene chloride, washed with water, dried and evaporated to dryness. This residue was crystallized from acetone- Skellysolve B hexanes to give 0.47 gram of 5a,llfl,l7atrihydroxy 6,8 fluoro 21 acetoxyall'opregnane-3,20- dione 3-ethylene ketal, melting point 220 to 228 degrees centigrade.

PREPARATION 7 A solution of 0.47 gram of 5a,11fl,l7a-trihydroxy-6/8- fiuoro 21 acetoxyallopregnane 3,20 dione 3-ethylene ketal in 35 milliliters of acetone and four milliliters of 1N sulfuric acid solution was gently boiled on the steam bath for ten minutes, cooled and neutralized with dilute sodium bicarbonate solution. Addition of water and cooling gave 0.33 gram of 5a,1l,B,l7a-trihydroxy-6fifluoro 21 acetoxyallopregnane 3,20 dione, melting point 230 to 240 degrees centigrade.

PREPARATION 8 fi-fluor0-I 1,8,1 7u-dihydroxy-2I-ace t0xy-4-pre gne te-3,20- dion e (6B-flu0rohydrocorfisone acetate) A solution of 100 milligrams of 5a,1l,6,17o-trihydroxy- 6/3-fluoro-21-acetoxyallopregnane 3,20-dione in 4.9 milliliters of acetic acid and 0.1 milliliter of water was refluxed for a period of one hour, cooled, diluted with fifty An additional 226 milligrams was obtaineds degrees centigrade.

milliliters of water and evaporated to dryness under reduced pressure. The residue was chromatographed over Flor-isil (synthetic magnesium silicate) to give one fraclion (77 milligrams) eluted with methylene chloride plus ten percent acetone. Crystallization from acetone- Skellysolve B hexanes gave 38 milligrams of 6fl-fluoro- 1 1,8, l7a-dihydroxy-21-acetoxy-4-pregnene-3,20-dione 6B- fluoro-hydrocortisone acetate), melting point 210 to 218 Infrareddata and ultraviolet data were found to be in agreement with the structure.

PREPARATION 9 lsom erization. of 6,8- to 6a-flu0r0hydr0c0rtis0ne acetate A solution of 0.132 gram of 6/3-fluorohydrocortisone acetate in twelve milliliters of chloroform and 0.1 milliliter of absolute alcohol was cooled to minus ten degrees centigrade in an ice-salt bath and a stream of anhydrous hydrochloricacid was gently bubbled through the solution for 2.5 hours while the temperature was maintained between minus five aud minus fifteen degrees centigrade.

The solution was then diluted with 25 milliliters of chloroform, washed with dilute sodium bicarbonate and water, dried over anhydrous sodium sulfate, and evaporated to drynessunder reduced pressure at sixty degrees Centigrade or less. Crystallization of the residue from acetone- Skellysolve B gave 42 milligrams of product, (Six-fluorohydrocortisone acetate, melting point 203 to 210 degrees Centigrade. I

PREPARATION 10 oa-fluoro-l 15,1 7ot,2] -trihydr0xy-4-pregnene-3,20-di0ne 6 u-fluorohydrocortisohe) .extract,;after drying over sodium sulfate, was chromatographed over a column of eighty grams of synthetic magnesium silicate.v The product fraction was. eluted with Skellysolve .B hexanes plus twenty and thirty percent acetone and gave 770 milligrams of 6a-fluorohydrocortisone which melted at 192 to 195 degrees centigrade after crystallization from ethyl acetate-Skellysolve Bhexanes. An

Jp'er minute to five liters of beer).

.incubation, when a good growth had been developed, five Analysis.Calculated for C H O F: C, 66.29; H, 7.68; F, 4.99. Found: C, 66.28; H, 7.65; F, 4.43.

PREPARATION 11 1 -dehydro 6a-fluorohydrocortisone Five 100-milliliter portions of a medium, in 250-milliliter Erlenmeyer flasks, containing one percent glucose, two percent corn steep liquor (sixty percent solids) and tap water, were adjusted to a pH of 4.9. This medium was sterilized for 45 minutes at fifteen pounds per square inch pressure and inoculated with a one to two day growth of Septomyxa afiinis, A. T.-C. C. 6737. The Erlenmeyer filask was shaken at room temperature (about 26 to 28 degrees centigrade) for a period of three days. At the end of this period this 500-milliliter volume was used as an inoculum for ten liters of the same glucosecorn steep liquor medium which in addition contained ten milliliters of an antifoam compound (a mixture of lard' oil and octadecanol). The fermentor was placed into the water-bath, adjusted to 28 degrees centigrade and the contents stirred (300 R. P. M.) and aerated (0.3 liter air After 24 hours of grams of 6a-fiuorohydrocortisone acetate plus one-half gram of 3-ketobisnor-4-cholen-22-a1, dissolved in 25 milliliters of dimethylformamide was added and the incubation carried out at the same temperature (28 degrees centigrade) and aeration for a period of 72 hours (final pH 8.3). The mycelium was filtered OE and washed with water. The wash'water was combined with the filtrate and the whole was extracted with three two-liter portions of a mixture of methylene chloride ethyl acetate (3:1). Removal of the solvent by evaporation gave 5.25 grams of crude solid which was triturated twice with four milliliters of methylene chloride to give 2.4 grams of l-dehydro-6u-fluorohydrocortisone of melting point 198 to 203 degrees centigrade. An analytical sample, recrystallized from acetone, melted at 202 to 204 degrees centigrade. Analysis gave [ocl plus 73 degrees (dioxane) and the following:

Analysis.Calculated for C H O F: C, 66.65; H, h

7.10; F, 5.02. Found: C, 66.69; H, 7.19; F. 5.49.

PREPARATION 12 6.95; F, 4.52. Found: C, 65.58; H. 7.16; F, 4.39.

PREPARATION 13 60c fluoro 17a hydroxy 21 acetoxy 1,4,9(11)- pregnatriene-3,20-di0ne To a solution of 1.05 grams of 1-dehydro-6a-fluorohydrocortisoue acetate in ten milliliters ,of pyridine was added 0.517 gram'of N-bromoacetamide. The mixture was allowed to stand under nitrogen for fifteen minutes, at which time it was cooled to five degrees centigrade. While stirring,'sulfur dioxide was passed over the surface until the solution gave no color change with acidified starch-iodide paper.

The temperature of the reaction mixture was not allowed to go above twenty degrees centigrade during the sulfur dioxide addition. The mixture was then poured into milliliters of ice-water, resulting in precipitation of 977 milligrams of 6a-fiuorol7a hydroxy 21 acetoxy 1,4,9(l1) pregnatriene- 3,20-dione, melting point 186 to 196 degrees centigrade (with decomposition). An analytical sample melted at 213 to 216 degrees centigrade (with decomposition). Analysis gave [ul plus 34 degrees (acetone) and the following:

Analysis.Calculated for C23H27O5FI C, 68.64; H, 6.76; F, 4.72. Found: C, 68.85; H, 6.86; F, 4.72.

PREPARATION 14 60c fluoro 9a bromo 116,170; dihydroxy 21 acetoxy 1,4 pregnadiene 3,20 dione (1 dehydro- 60c fluoro 9oz bromohydracortisone acetate) To a solution of 1.27 grams of 6ot-flll0IO-17a-hYdIOXY- 21 acetoxy 1,4,9(11) pregnatriene 3,20 dione in 19.5 milliliters of methylene chloride was added 38 milliliters of tertiary butyl alcohol, a solution of three milliliters of 72 percent perchloric acid in 22.5 milliliters of water, and a solution of 0.55 gram of N-bromoacetamide in 9.6 milliliters of tertiary butyl alcohol. After stirring for fifteen minutes, a solution of 0.55 gram of sodium sulfite in thirty milliliters of water was added and the mixture concentrated under reduced pressure at sixty degrees centigrade until crystallization occurred. After cooling in an ice bath, 100 milliliters of water was added with stirring. On filtering the crystalline product, followed by washing with watcr and drying, a yield of 1.59 grams of essentially pure 6oz-fill010-9a-bIOI'I10-ll/3, 17a dihydroxy 21 acetoxy 1,4 pregnadiene 3,20- dione, melting point 188 to 191 degrees centigrade (with decomposition) was obtained. The product can be used in the succeeding example without further purification.

PREPARATION 15 6a fluoro 913,115 oxido 17oz hydroxy 21 acetoxy- 1,4-pregnadiene-3,20-dione A mixture of 1.749 grams of 60c-fll1010-9a-b1'01110-11fi, 17oz dihydroxy 21 acetoxy 1,4 pregnadiene 3,20- dione (1 dehydro 6oz fluoro 9oz bromohydrocortisone acetate), 1.749 grams of potassium acetate, and fifty milliliters of acetone was stirred and heated at reflux temperature for eighteen hours. The reaction mixture was then concentrated to about one-half the original volume, cooled and poured into 300 milliliters of water to give 1.303 grams of 6a-fiuoro-913,1l 8-oxido-17a-hydroxy- '2l-acetoxy-1,4-pregnadiene-3,ZO-dione, melting point 234 to 238 degrees centigrade (with decomposition). An analytical sample, recrystallized from acetone, melted at 257 to 260 degrees centigrade. Analysis gave [011 plus seventy degrees (acetone) and the following:

Analysis.--Ca1culated for C H O F: C, 66.01; H, 6.50; F, 4.54. Found: C, 65.73; H, 6.58; F, 3.87.

PREPARATION 16 6a,9a difluoro 116,170; dihydroxy 21 acetoxy 1,4- pregnadiene 3,20 dione (1 dehydro 6a,9a difluorohydrocortisone acetate) To 5.2 grams of liquid hydrogen fluoride cooled in a Dry-Ice bath, was added, portion-wise, a slurry of 2.276 grams of 6a-fluoro-9B,11,8-oxido-17u-hydroxy-21-acetoxy- 1,4-p1'egnadiene-3,20-dione in nine grams of tetrahydrofuran (distilled over NaOH) and 28 milliliters of methylene chloride which had similarly been cooled in a Dry- Ice bath. The steroid dissolved completely. After standing at zero to five degrees centigrade for seventeen hours, the reaction mixture was poured slowly into a stirred mixture of 500 milliliters of water and 25 grams of sodium bicarbonate. The mixture was stirred for a few minutes, and the product was extracted with three 100-milliliter portions of methylene chloride. The meth- The fraction eluted from tl ecolnmn with fifteen and twenty percent acetone in Skellysolve B hexanes was recrystallized from ethyl acetate-Skellysolve B hexanes and gave 1.342 grams of 1-dehydro-6a,9a difluorohydrocortisone acetate, melting point 238 to 242 degrees centigrade. An analytical sample melted at. 239 to 242 de .grees centigrade. Analysis gave plus 91 degrees (acetone) and the following:

Analysis.-Calculated for C H O F C, 63.00; H,

6.4-4; F, 8.67. Found: C,'63.23; H, 6.82; F, 8.14

= PREPARATION 17 601,90: difluoro 11B,]7oc,21 trihydroxy 1,4 pregnadi ene 3,20 dione (1 dehydro- 60,9a difluorohydrocortisone) Nitrogen was bubbled through a solution of 1.4 grams of 611,904 difiuoro llfi,l7ot.- dihydroxy 21 acetoxy- 1,4 pregnadiene 3,20 dione (1 dehydo 60,906 di- -fiuorohydrocortisone acetate) in 140 milliliters of methanol for fifteen minutes. To this was added a solution of 1.4 grams of potassium bicarbonate in 175 milliliters of water, likewise treated with nitrogen. After stirring under nitrogen for five hours, the base was neutralized by the addition of 1.5 milliliters of acetic acid in forty milliliters of water. The mixture was then concentrated under reduced pressure at 55 degrees centigrade analytical sample melted at 250 to257 degrees centigrade (with decomposition). Analysis gave [041 plus 84 degrees (acetone) and the following;

Analysist--Calculated for C21H25O5F2LC, 63.62; H, 6.61; F, 9.59. FoundzC, 62.26: H, 7.10; F,-9.41.

EXAMPLE 1 fiuoro-l 1,3,1 6a,] 7a,214etrcvhydroxy-l,4-pregna1diene- 3,20-di0ne One hundred milliliters of two percent cornsteep liquor of sixty percent solids was adjusted to pH of 6.8 to 7.4 with sodium hydroxide and was sterilized at fifteen pounds pressure for thirty minutes; --.To this was added a similarly sterile solution of two grams of Cerelose (technical grade of dextrose) in four milliliters of Water. This sterile medium was inoculated with a suspension of spores and mycelium of Streptomyces roseochromogenus (Waksman Collection No. 3689) and was agitated on a rotary shaker for a period of 24 hours by which time a good growth of the organism had taken place. To this 24 hour culture was added twenty milligrams of 6afluoro 11fi,170,21 trihydroxy 1,4 pregnadicne 3,20- dione (1-dehydro-6m-fluorohydroc0rtisone) dissolved in 0.2 milliliter of dimethylformamide. Incubation of the steroid was maintained (with agitation) for five days, at which time the pH was 8.6. The fermentation broth was then separated into the mycelium and the beer by centrifugation. The mycelium was extracted first with two 25- milliliter portions of acetone and then with four successive 25-milliliter portions of methyl isopropyl ketone. The beer was extracted with four successive 25-milliliter portions of methyl isopropyl ketone. All of the extracts were combined, washed with two percent aqueous sodium bicarbonate solution and with water, dried with an hydrous sodium sulfate, and evaporated to dryness. The residue, which on paper chromatogram analysis showed the presence of 6u-fluoro11,8,16a,Hall-tetrahydroxy- 1,4-pregnadiene-3,20-dione, was purified by chromatography over synthetic magnesium silicate (Florisil) and crystallization from acetone to give 6a-fluoro-ll,8,l6a, 17a,2l-tetrahydroxy-1,4-pregnadiene-3,20-dione.

Following the procedure of Example 1 above, but substituting 1-dehydro-6u,9u-difluorohydrocortisone as starting material therein, is productive of 6a,9oc-difluoro-11fl, 16oz, l7rx,21-tetrahydroxy-1,4-pregnadiene-3 ,20-dione.

EXAMPLE 2 6 a-fluoro-l 1 13,1 6 0a,] 70,21 -tetrahydr0xy-1,4-pregnadiene-3, ZO-dione 16,21-a'iacetate A solution of 1.2 grams of 6zx-fluOrO-1lfl,l6rx,l7a,2ltetrahydroxy-1,4-pregnadiene-3,20-dione (from Example 1) in twenty milliliters of pyrodine' and twenty milliliters of acetic anhydride was allowed to stand-at room temperature (about 25 degrees centigrade) for eighteen hours and was then poured into 200 milliliters of ice-water. The resulting rnixturewas extracted with methylene chloride and the extract was washed with dilute hydrochloric acid, dilute sodium bicarbonate, and water. After drying the solution with anhydrous sodium sulfate, the solvent was removed by evaporation and the residue was purified by chromatography over synthetic magnesium silicate (Florisil) and crystallization from acetone to give 6ot-fluoro-1113,16a,17a,21-tetrahydroxy-1,4-pregnadiene-3,20-dione 16,21-diacetate.

Following the procedure ofExarnple 2 above,butsubstituting 6 ,9ct -difluoro-116,16a,17u,21-tetrahydroxy-1,4- pregnadiene-3,20-dicne as starting material therein is productive of6a,9ot-difiuoro-11B,16a,17u,21-tetrahydroxy- 1,4-pregnadiene-3 ,20-dione 16,21-diacetate.

Similarly, acylation of 6a-fiuoro-11fi,16a,17a,21-tetrahydroxyd,4-pregnadiene-3,20-dione with the appropriate acid anhydride or acid chloride is productive of still other 16,21-diacylates, such as for example, 6a-fiuoro-11/3,16a,

'17a,2l-tetrahydroxy-l,4rpregnadiene-3,ZO-dione 16,21-dipropionate, 600- fluoro- 11fl,16a,17at,21 tetrahydroxy- 1,4-pregnadiene-3,20-dione 16,21-dibutyrate, 6a-fluoroll/i,l5oc,l7ot,2l tetrahydroxy 1,4 pregnadiene 3,20-

droxy-l,4-pregnadiene-3,20 dione 16,21-dihexanoate, 60ciluoro 11,8,16oc,l7ot,21 tetrahydroxy 1,4 pregnadiene- -3,20-dione 16,2 l-dilaurate 6m-fluoro-l 1p,16a,17a,21-tetrahydroxy- 1 ,4-pregnadiene-3 ,20-dione 16,21-di-(trimethyl acetate), 6a-fluoro-11,6316,17a,21-tetrahydroxy-1,4-preg- 17t,2l-tetrahydroxy-l,4-pregnadiene-3,20-dione 16,21-diisovalcrate, 60c fiuoro 7 115,16a,17a,21 tetrahydroxy- 1,4 pregnadiene-3,20rdione 16,21-dicyclohexane carboxylate, 60:" fiuoro 11,8,16a,17u,21 tetrahydroxy 1,4-

50 pregnadiene-3,20-dione 16,21-dibenzoate, 6a-fluoro-11B,

16a, 17ot,2l-tetrahydroxy-l,4-pregnadiene-3,ZO-dione 16, 21 diphenylacetate, 6a fluoro 11/3,16a,17a,21 tetrahydroxy 1,4 prcgnadiene 3,20 dione 16,21 di (,8- phenyl) propionate, 60c fiuoro 11e,16m,17a,21 tetrahydroxy-l,4-pregnadiene-3,20-dione 16,21-di-(o-, m-, ptoluate) 6a-fluoro-115,16a,170:,21-tetrahydroxy-1,4-pregnadiene-3,20-dione 16,21-dihemisuccinate, 6ct-fiuoro-11B, 16m,17a,2l-tetrahydroxy-1,4-pregnadiene-3,20-dione 16, ZI-dihemiadipate, 6u-fluoro-11B,16u,17u,21-tetrahydroxy- 1,4-pregnadiene-3,20-dione 16,21-diacrylate, 60L-fil10l'0- 11,8,16a,17a,21 tetrahydroxy 1,4 pregnadiene 3,20- dione 16,2l-diundecylenate, 6oLfi1lOIO-ll,8,l6cc,l7ot,2ltetrahydroxy-1,4-pregnadiene-3,20-dione 1 6,21-dipropiolate, 6a-fiuoro-11 8,16a,17a,21-tetrahydroxy-1,4-pregnadiene-3,20-dione 16,21-dicinnamate, 6KX-fillOI'O-11[3,16OL, 17,2l-tetrahydroxy-1,4-pregnadiene-3,20-dione 16,21-dimaleate, 6ot-fluoro-11,8,16a,17a,21-tetrahydroxy-1,4-pregnadiene-3,20-dione 16,21-dicitraconate, and the like.

Similarly, acylation of 6a,9a-difluoro-11B,16a,17u,21- tetrahydroxy-1,4-pregnadiene-3,20-dione with the appro priate acylating agent is productive of the corresponding 16,21-diacylates. The preferred diacylates are those corresponding to the diacylates described above for 6wfluoro 11/3,16u,17ot,21 tetrahydroxy 1,4 pregnadiene- 3,20-dione.

minu at, ,v ."(4

I tetrahydroxy-l,4-pregnadiene-3,ZO-dione 7 ing ll-keto 16,21-diester compounds.

EXAMPLE 3 triorte 16,21-diacetate To a solution of 0.5 gram of 6oc-flu01'O-l1B,16oc,l7ot,2l-

l6 ,2l-diacetate in twenty milliliters of acetic acid was'added a solution of 0.15 gram of chromium trioxide in one-half milliliter of water.

period of four hours. Thereafter the excess oxidant was 7 destroyed by the addition of 0.5 milliliter of methanol and the mixture was poured into 100 milliliters of water and extracted with methylene chloride. The extract was washed with dilute sodium bicarbonate and with water, and was dried and evaporated to dryness. The residue was crystallized from acetone to give 6a-fluOI016oc,17oz, 21-trihydroxy-1,4-pregnadiene-3,11,20-trione 16-21-diacetate.

Following the procedure of Example 3 above, but subpregnadiene-3,20-dione 16,21-diacetate as starting material therein is productive of 6zx,9a-diflll0l0-16oz,17a,21-t1ihydroxy-1,4-pregnadiene-3,11,20-trione 16,21-diacetate.

Similarly, oxidation of other llfi-hydroxy 16,21-diester compounds of Example 2 is productive of the correspond- The preferred 11- keto 16,21-diester compounds are those wherein the acyl radical is that of an organic carboxylic acid, preferably a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

EXAMPLE 4 6 u-fluoro-J 6 0a,] 704,21 -trihydr0xy-1 ,4-pregnadiene-3 ,1 1,20-

trione A solution of 1.2 grams of 6ot-fluoro-16a,17a,21-trihydroxy-l,4-pregnadiene-3,l1,20-trione 16,21-diacetate, two grams of potassium bicarbonate, 100 milliliters of methanol and fifteen milliliters of water was purged with nitrogen and stirred at 25 degrees centigrade for eight hours. The solution was then neutralized by addition of acetic acid and the methanol was removed by distillation under reduced pressure. The residue was extracted with 100 milliliters of methylene dichloride and the extract,

after drying over sodium sulfate, was chromatographed over a column of eighty grams of synthetic magnesium silicate. The product fraction was eluted with Skellysolve B hexanes plus thirty percent acetone and gave 6w fluoro 16a,17a,2l trihydroxy -.1,4-pregnadiene-3,11,20- trione which was recrystallized from acetone.

The mixture was stirred and maintained at room temperature (about 25 degrees centigrade) for a -Following-the procedure of Example 4,"- saponification 3,11,20-trione 16,21-diacetate of Example 3'is productive of 6a, 9a-difiuoro-16a,17u,21-trihydroxy-1,4-pregnadiene- 3,11,20-trione.

It is to be understood that the invention is not to be limited to the exact details of operation or exact compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the scope of the appended claims. We claim: 1. A 16-oxygenated pregnadiene CH3 7 M (llHzoR I I C=0 ---0n of the formula.

. wherein Y is selected from the group consisting of hydrogen and fluorine, X is selected from the group consisting of the carbonyl radicaland the B-hydroxymethylene radical, and R is the same member selected from the group consisting of hydrogen and the acyl radical of a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

2. 6a-fluoro-1118,16a,17a,21tetrahydroxy-1,4-pregnadiene-3,20-dione.

No references cited.

Notice of Adverse Decision in Interference In Interference N 0. 90,125 involving Patent N 0. 2,838,546, B. J. Mag

W. P. Schneider, 0. K. Sebek and G. B. bpero, Ga-FLUORO-lGa-HYDROXY- IA-PREGN ADIEN ES, final judgment advers Apr. 30, 1964, as to claims 1, 2, 3, 4, 5, 6,7, 8 and 9 [Ofiicz'al Gazette May 4, 1965.] 

1. A 16-OXYGENATED PREGNADIENE OF THE FORMULA 